利用Tet—on可诱导系统构建稳定表达PESl的SKOV3细胞株

Establishment of ovarian SKOV3 cell line stably expressing PESI by Tet-on inducible system

目的为了进一步研究PES1的生物学功能，采用Tet—on系统构建可诱导稳定表达PES1的卵巢癌细胞株SKOV3。方法采用PCR技术将PES1克隆到pTRE—Tight载体中，并进行表达鉴定。将调控质粒pTet—on转染卵巢癌细胞，经G418筛选后再转染pTRE—Tight—PES1，然后经潮霉素筛选出阳性克隆。用不同浓度的强力霉素（Dox）进行诱导后，Westernblot确定Dox的最佳诱导浓度。结晶紫实验观察Dox诱导的稳定转染pTRE—Tight-PES1的SKOV3细胞生长速度。结果将成功构建的pTRE—Tight—PES1转染SKOV3细胞后，经过筛选获得Dox可诱导表达PES1的SKOV3细胞克隆。浓度低于2mg／L的Dox可以剂量依赖性地诱导PES1表达，2mg／L可以诱导PES1高表达。Dox诱导的转染pTRE—Tight和未转染任何质粒的SKOV3生长速度无明显差异，而转染pTRE—Tight—PES1的SKOV3细胞比转染pTRE-Tight和未转染任何质粒的细胞在第g天时生长速度明显更快（P=0．001）。结论成功建立了可诱导表达PES1的SKOV3细胞，为研究PES1的生物学功能提供了有效的细胞模型。PES1表达可以增强SKOV3细胞的生长。

Objective To further research the biological functions of PES1, the ovarian SKOV3 cell line with inducible stable PES1 expression is established by using Tet-on system. Methods PES1 was cloned into pTRE-Tight vector via PCR and its expression was identified. After transfected the regulating plasmid pTet-on, SKOV3 cells were screened with G418 and re-transfeeted pTRE-Tight-PES1. The positive cell clones were screened out with hygromycin and were induced by doxyeycline （Dox） to definite the best induction con- centration. Growth velocity of SKOV3 cells stably expressing PES1 induced by Dox was detected with viola erys- tallina. Results The SKOV3 cells with inducible PES1 expression were screened out after the cells were trans- fected pTRE-Tight-PES1 constructed. Dox could dose-dependently induce the PES1 expression with the concen- tration under 2 mg/L, and 2 mg/L of Dox induced the highest PES1 expression. Growth velocity of SKOV3 cells transfected pTRE-Tight has no significant difference between the SKOV3 cells transfected nothing induced with Dox. However, the SKOV3 cells transfected pTRE-Tight-PES1 grew faster than the cells transfected pTRE- Tight or without transfection in the fourth day （ P = 0. 001 ）. Conclusion The inducible stable PES1 expres- sion SKOV3 ceils are successfully established and could be used to be an effective cell model to research the biological functions of PES1. The expression of PES1 could promote the growth of SKOV3 cells.